Synthetic double-helical polynucleotide

While the complementary double helical structure explained how particular sequences of bases could be used to store a genetic instruction it was not immediately clear how replication occurred or, indeed, how these instructions were used. Later work by Gamow linked DNA base pair sequences to protein synthesis [15] but it was not until 1961, when Nirenberg and Matthaei demonstrated that cell-free protein synthesis relied upon synthetic or natural polynucleotides [16], that the final link was made. The information held within the linear DNA sequence is replicated every time a cell divides. Replication is possible because of the unique double helical structure of DNA as shown in Fig. 2.7. 

One well-established observation is that, under conditions where single-stranded polynucleotides give rise to a d.c. polarographic reduction wave, both native DNA and other double-helical natural and synthetic polynucleotides are inactive 22 23,46-47, 58,59,61) Tjjjs js rea(ji]y interpretable in that, in such helical structures, the adenine and cytosine residues are located in the interior of the helix, and hydrogen bonded in complementary base pairs (see below). Z-DNA, in which cytosine residues are at the surface of the helix, is of obvious interest in this regard, and the B - Z transition in the synthetic poly(dG dC) has been investigated with the aid of differential pulse polarography and UV spectroscopy 60). 

Until recently, only right-handed double helices were known. A left-handed helix structure was observed for the first time in the synthetic hexameric DNA containing alternating cytosine and guanine bases [d(CGCGCG)]. Both polynucleotide strands are wound around themselves in a left-handed sense. The phosphate groups show a zick-zack-type pattern along the screw this structure therefore was named Z-DNA (Fig. 2). 

Since RNA double helices and most RNA/DNA hybrids have only been observed in conformations similar to the A-form [14-24] whereas studies of the DNA conformation in cells such as salmon sperm have revealed that the resting state of DNA appears to be the B-form [25], it is natural to speculate that the B-form is adopted for DNA replication whereas the A-form is adopted for transcription. Similar considerations apply in the case of synthetic polynucleotides a polynucleotide with a highly repetitive base sequence can be found in the S form under conditions in which a natural DNA with essentially random base sequence would be found in the B form. Highly repetitive sequences flanked on both sides by random sequences are known to exist in natural DNAs and so it is possible that under some ionic conditions the repetitive and random sequences would have different conformations. The potential to exploit such structural differences in control processes mediated by specific DNA-protein recognition mechanisms is clear. Although these speculations have not been confirmed, the fact that conformational transitions may be implicated in such fundamental biological processes is powerful justification for extensive study of the stereochemical pathways of these transitions and the factors which promote and control them. 

The anti-poly I poly C antibodies react not only with RNA of reovirus but also, though to a lesser degree, with RNA extracted from mammalian cells (Fig. 4). Comparison of the efficiency of inhibition of the cross-reaction with mammahan RNA by RNA of reovirus, by double-helical complexes of synthetic polynucleotides and by single-stranded polynucleotides, has shown that the antibodies anti-poly I poly C react especially with double-stranded conformational determinants of the RNA. None of the single-stranded polynucleotides is capable of totally inhibiting the reaction with RNA whatever... 

Differences in the capacity of inhibition by polynucleotides not involved in complementary hydrogen bonds and by double-helical complexes of synthetic polyribonucleotides, or double-stranded viral RNA allow the conclusion that it is above all the regions of associated base pairs which are recognized in the RNA by anti-poly I poly C antibodies. Such complementary double-stranded helical regions have been described especially in tRNA but they have also been shown to exist in ribosomal RNA. These two kinds of RNA were therefore isolated and studied separately. Although both fractions precipitate anti-poly I poly C antibodies, their reactivity is nevertheless very different and rRNA precipitates eight times as much antibody as tRNA. Since tRNA possesses an important tertiary structure, this low reactivity could be explained by the non-accessibility of antigenic sites. 

It is essentially the cross-reactions with another double-helical complex, poly A poly U, which have been studied with immune sera of mice and hamsters. These cross-reactions have been observed very frequently with immune sera of RAP mice and B/W mice and to a lesser degree with the sera of hamsters poly A poly U is the best inhibitor of the reaction between anti-poly I poly C of mice and the homologous antigen (Lacour et al., 1971 Steinberg et al., 1971). The role of the bases in this immunoreaction does not appear to be essential. It is probable that, as in rabbit, these antibodies recognize double-helical structures. While there is similarity in the reactions of the sera of the three species with synthetic polynucleotide double-helical complexes, the cross-reactions of the anti-poly I poly C antibodies with nucleic acids are very different in the rabbit, the mouse, and the hamster (Table 4). 

Just as main-chain NH 0=C hydrogen bonds are important for the stabilization of the a-helix and / -pleated sheet secondary structures of the proteins, the Watson-Crick hydrogen bonds between the bases, which are the side-chains of the nucleic acids, are fundamental to the stabilization of the double helix secondary structure. In the tertiary structure of tRNA and of the much larger ribosomal RNA s, both Watson-Crick and non-Watson-Crick base pairs and base triplets play a role. These are also found in the two-, three-, and four-stranded helices of synthetic polynucleotides (Sect. 20.5, see Part II, Chap. 16). 

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